We are studying the mechanisms that maintain constitutive heterochromatic regions in a condensed form. We earlier investigated the role of Argonaute 2 (Ago2) in generation and maintenance of a heterochromatic domain. We have now investigated the binding of Ago2 to the ribosomal gene repeat locus in the HEK293T cell line. We find that there are chromatin binding sites for AGO2 throughout the 45S region of the human rRNA gene, reflecting Ago2 interaction with sites on the nascent 45S RNA transcript. The interactions are specific,mediated by binding of microRNAs to the RNA. Knockdown of Ago2 resulted in an increase in rRNA expression, suggesting a regulatory role for Ago2 within the nucleolus. In other studies addressing the structure of heterochromatin, we have initiated experiments to determine the three dimensional genomic organization of non-ribosomal genes within human nucleoli. This is a comparative study involving nucleoli from a variety of cell lines. Early results show that there are considerable differences in genomic organization among cell types.